Cornerstone® Biomanufacturing Development Services for other viral particles and VLPs offer comprehensive solutions, including BSL1 and BSL2 labs for non-GMP production. Utilizing both adherent and suspension cells, the services focus on optimizing transfection/infection and production processes, with bioreactor scale-up capabilities to 10 liters. Emphasis is placed on DSP-focused optimization to ensure a holistic approach, enhancing overall process efficiency and effectiveness.
In downstream processing, services cover all BSL1 and BSL2 viral particles, VLPs, and inactivated particles, including Influenza, NDV, Vaccinia, Lenti, Pox, KHV, chikungunya, and Lassa, as well as VLPs derived from different USP. Options for single-step or full processes are available, ensuring thorough and effective production. Detailed reports with results and SOPs are provided to support seamless integration and success in production endeavors.
Upstream Process Development
Downstream Process Development
Upstream Process Development
Downstream Process Development
This phase often demands significant time and resources, involving products such as cell lines, media and various other reagents from producers with different licensing agreements and royalties, driving up costs.
The experienced team optimizes product combinations for peak performance, saving development time. A streamlined process is available using Sartorius raw materials.
Frequently the viral seed used in early phases is not highly concentrated nor pure, creating challenges in reproducibility and scale-up.
Before starting, parameters affecting transfection or infection are thoroughly optimized, including cell density, quantity of DNA, ratio between DNA and transfection reagent, MOI, feed, induction, duration of production, and other complex formation parameters.
The smallest scale in adenovirus production is typically 2L. Conducting parallel experiments at this scale requires a significant investment in multiple 2L vessels or more time, increasing variability between conditions. Total volumes escalate quickly, driving up costs.
The Ambr® 250 modular system, with 6 single-use vessels operating in parallel, is utilized to reduce variability and accelerate optimization. Processes developed at this scale are scalable across the entire Sartorius portfolio, saving up to 4-5 times in raw materials compared to the 2L scale.
Process analytical technology (PAT) is employed to ensure optimal conditions for the entire process development. Factors such as titer and impurities are crucial not only in the upstream process but also impact downstream processing, influencing the overall success of the bioprocess.
Frequently, DSP optimization begins only after the upstream process USP is established, limiting the ability to adjust both bioprocess parts in tandem.
From the smallest scales, USP is optimized to enhance DSP and reduce costs. Scaling up to 5L or 10L allows for the production of sufficient material to test the entire process using scalable products. The entire bioprocess can be performed without freezing material between USP and DSP for shipment, closely mimicking GMP conditions.
If the lysis step is needed it is developed in regards to dependency on the chromatography purification that follows or vice versa. Consequently, the ratio of the components in the lysis buffer has to be adapted and the optimum has to be found.
To define optimal lysis conditions, a holistic approach based on accumulated experience is employed. Different combinations of components in the lysis buffer are tested with subsequent steps in mind, optimizing lysis to align with the following steps and focusing on achieving the highest yield.
To clarify the lysate several steps of filtration are used, firstly coarse and then fine filtration. Sartorius filters with cut off 3, 0.8 and 0.45 μm are used. Expected issues are related to sheer stress to which the virus is exposed and filtration scale-up.
Each filtration step is optimized to achieve a step recovery close to 100%. If the starting material does not behave as expected, additional steps are incorporated into the clarification process. The filtration scale-up is meticulously planned and tested, provided the material quantity permits.
Sometimes TFF needs to be implemented. With ultrafiltration and diafiltration we partially purify the product and minimize the volume of material. Issues can occur if the material is not clarified enough, if the membrane area is not sufficient, the procedure is time-consuming and product is exposed to sheer stress.
The ultrafiltration and diafiltration steps are ensured to be carried out correctly by carefully analyzing various parameters, conducting small-scale tests, and drawing on previous experiences.
The decision on the cleaning steps is influenced by the application, the initial material, customer requirements and other factors. The two most common options are noted in the scheme. Challenges are not expected in this phase.
The steps for the final product are optimized to meet the required criteria and ensure alignment with customer demands.
The final formulation step can be part of the chromatographic step otherwise it is a separate step. If the purification process does not allow combining chromatography and formulation in a single step, an additional chromatographic step or UF-DF is required.
The process is developed with a minimum number of steps to prevent product loss, taking into account clients’ needs, available equipment, and timelines.
In downstream processing, a range of analytical methods is employed to ensure quality and efficiency. The PATfix analytical platform is used for detecting and semi-quantifying the product, as well as monitoring host cell-related impurities released during cell lysis. Additionally, d/ddPCR is utilized for quantitative analysis of viral genome (vg) titers. A cell-based infectivity assay is conducted to assess infectious titer, serving as a crucial analytic to select conditions that do not affect product infectivity. Total protein and total DNA are also monitored.
In summary, each analytical method mentioned is used to measure target molecules and critical impurities, integrating the steps of downstream processing into a coherent and successful process.
If the lysis step is needed it is developed in regards to dependency on the chromatography purification that follows or vice versa. Consequently, the ratio of the components in the lysis buffer has to be adapted and the optimum has to be found.
To define optimal lysis conditions, a holistic approach based on accumulated experience is employed. Different combinations of components in the lysis buffer are tested with subsequent steps in mind, optimizing lysis to align with the following steps and focusing on achieving the highest yield.
Any virus, VLP or other similar structure can be purified with monoliths. There are different chemistries available, that can serve as a chromatographic tool for purification. Every product of interest has its own features and characteristics, which we exploit when developing the purification method. In theory, this means that there is no uniform process for all viruses, VLPs, and similar structures and the method is developed as a custom made. Different viruses have been tested so far: Influenza, NDV, Vaccinia, Lentivirus, Pox, KHV, chikungunya, Lassa… as well as VLPs derived from different USP.
It depends on the application, but to start with, we recommend CIMmultus QA, SO3 and OH.
Yes, different forms of the same virus can be separated. It is not a straightforward method and it requires some in-depth research and experiments, but such kind of methods can be developed.
Different orthogonal analytics are used for different applications. In general, we tend to have at least three independent analytical methods to assure us seeing the real picture of the process.
Our experts would be happy to discuss your project. Fill the contact form below or send us an email to support@biaseparationsinc.com
Your inquiry is empty